Injectable organic composite hydrogel combined with photo-thermal chemotherapy as well as preparation method and application of injectable organic composite hydrogel

Abstract

The invention belongs to the technical field of nano biological medicines, and particularly relates to injectable organic composite hydrogel combined with photo-thermal chemotherapy as well as a preparation method and application thereof. The injectable organic composite hydrogel combined with photo-thermal chemotherapy is composed of an orthoester compound and a water-phase hydrogel, the orthoester compound is emulsified and then uniformly dispersed in the hydrogel, and the hydrogel is formed by crosslinking carboxymethyl chitosan and glutaraldehyde. In the injectable organic composite hydrogel combined with photo-thermal chemotherapy provided by the invention, firstly, oily orthoester is utilized to dissolve a cis-platinum prodrug and indocyanine green to obtain an orthoester compound, then the orthoester compound is uniformly emulsified and loaded in the hydrogel, dynamic imine bonds of the hydrogel respond to tumor slightly acidic environment breakage, and then the dynamic imine bonds of the hydrogel are separated from the dynamic imine bonds of the hydrogel, so that the injectable organic composite hydrogel combined with photo-thermal chemotherapy can be used for preparing the injectable organic composite hydrogel combined with photo-thermal chemotherapy. The orthoester compound successfully escapes, along with degradation of the oily orthoester, the cis-platinum prodrug and the indocyanine green are self-assembled through hydrogen bonds to form nanoparticles, the enrichment and retention capacities of the two drugs at the tumor site are enhanced, and the overall curative effect is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano biomedical technology, and specifically relates to an injectable organic composite hydrogel for combined photothermal chemotherapy, a preparation method thereof, and an application thereof. Background Art

[0002] Due to the limitations of traditional treatment methods such as conventional chemotherapy or molecular targeted therapy, including poor drug enrichment ability, strong toxic and side effects, easy induction of tumor cell drug resistance, and poor treatment effects, although local drug delivery treatments such as suppositories and emulsions have achieved certain results, there are still great challenges to overcome these obstacles and improve clinical efficacy. Nano composite hydrogels have currently attracted wide attention because they possess the dual advantages of hydrogels and nano drugs, such as high tissue adhesion, and the enhanced permeability and retention effect of nanoparticles. However, nanoparticles have poor solubility in hydrogels, are prone to precipitation, are difficult to efficiently escape, and are easily cleared by the body after escape, unable to reach tumor tissues and play a role, resulting in limited overall treatment effects. Therefore, it is particularly important to enhance the efficient escape of drugs from hydrogels and enhance the tumor enrichment of drugs.

[0003] Related reports indicate that aqueous hydrogels can encapsulate oil-phase orthoester complexes to form organic composite hydrogels. Due to the immiscibility of oil and water in organic composite hydrogels, the oil phase is prone to escape from the aqueous hydrogel, which is beneficial to drug release. The cross-linking components of the aqueous hydrogel have good tissue adhesion, while reducing the interaction with tissues, facilitating the disintegration of the organic composite hydrogel. However, existing organic composite hydrogels have defects such as difficult drug release, poor drug enrichment and retention ability at the tumor site, and poor curative effects. Summary of the Invention

[0004] Aiming at the deficiencies of the above-mentioned existing technologies, the present invention provides an injectable organic composite hydrogel for combined photothermal chemotherapy, a preparation method thereof, and an application thereof. The injectable organic composite hydrogel for combined photothermal chemotherapy of the present invention is composed of an orthoester complex and an aqueous hydrogel. The orthoester complex is loaded with the chemotherapeutic drug cisplatin prodrug and the photothermal agent indocyanine green dissolved therein. The oily orthoester enhances the photothermal efficacy and stability of the photothermal agent. At the same time, due to the immiscibility of oil and water in the injectable organic composite hydrogel for combined photothermal chemotherapy, it promotes the effective release of the two drugs, cisplatin prodrug and indocyanine green. The oily orthoester assists the two drugs to penetrate at the tumor site, and after the degradation of the oily orthoester, it assists the two drugs to self-assemble into nanoparticles, comprehensively enhancing the tumor enrichment and retention of the two drugs. The chemotherapeutic drug cisplatin prodrug responds to the tumor microenvironment and is reduced to exert its effect, reducing systemic toxicity and achieving a strong tumor suppression effect of precise coordination of hyperthermia and chemotherapy, overcoming the technical defects of existing organic composite hydrogels.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention protects a preparation method of an injectable organic composite hydrogel for combined photothermal chemotherapy, comprising the following steps: Under an inert atmosphere, using anhydrous acetonitrile as a reaction solvent, mixing 3-amino-1,2-propanediol with ethyl trifluoroacetate, and carrying out an amidation reaction in the liquid phase. After the reaction is completed, the solvent is separated by rotary evaporation, extracted, dried, and rotary evaporated to obtain an intermediate product; using anhydrous acetonitrile as a reaction solvent, mixing the intermediate product with trimethyl orthoformate, and carrying out an acetalization reaction in the liquid phase under catalytic action. After the reaction is completed, the solvent is separated by rotary evaporation, extracted, dried, and rotary evaporated to obtain an oily orthoester; the oily orthoester can fully dissolve two drugs, cisplatin prodrug and indocyanine green, enhance the photothermal efficacy and stability of the photothermal agent, is insoluble in oil and water in the injectable organic composite hydrogel for combined photothermal chemotherapy, assists in accelerating the release of the two drugs, and at the same time, the oily orthoester has lipophilicity and acid sensitivity, can assist in enhancing the penetration of the two drugs at the tumor site, and degrades in the mildly acidic tumor microenvironment, assisting the two drugs to form nanoparticles through hydrogen bond self-assembly.

[0006] Mix divalent cisplatin in water, then dropwise add H2O2 solution, stir and carry out an oxidation reaction in the dark, separate the solvent, and lyophilize to obtain a tetravalent cisplatin product; using anhydrous N,N-dimethylformamide as a reaction solvent and triethylamine as a catalyst, mixing the tetravalent cisplatin product, norcantharidin, and triethylamine, reacting in the dark under nitrogen protection, and carrying out an esterification reaction in the liquid phase. After the reaction is completed, the solvent is separated by rotary evaporation, sedimented, washed, the precipitate is collected and dried to obtain a cisplatin prodrug; the cisplatin prodrug and its preparation method are prior arts.

[0007] Ultrasonically disperse the cisplatin prodrug and the photothermal agent indocyanine green in the oily orthoester to obtain an orthoester complex, then add a 2% carboxymethyl chitosan solution, vortex to emulsify the orthoester complex evenly, and add a 5% glutaraldehyde solution dropwise while vortexing to obtain an injectable organic composite hydrogel for combined photothermal chemotherapy. The injectable organic composite hydrogel for combined photothermal chemotherapy is composed of an orthoester complex and an aqueous hydrogel, which has a tight surface structure, an appropriate pore structure. The use of the small molecule crosslinking agent glutaraldehyde avoids the formation of unnecessary intermolecular forces with biological tissues, is beneficial to the release of the orthoester complex, and the introduction of carboxymethyl chitosan endows the injectable organic composite hydrogel for combined photothermal chemotherapy with good tissue adhesion. The amino group of carboxymethyl chitosan crosslinks with the aldehyde group of glutaraldehyde to form dynamic imine bonds, making the injectable organic composite hydrogel for combined photothermal chemotherapy have excellent injectability and self-healing performance.

[0008] Preferably, the conditions for the amidation reaction are: stirring at room temperature for 10 h to 15 h.

[0009] Preferably, the molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1 to 3.

[0010] Preferably, the conditions for the acetalization reaction are: stirring at room temperature for 10 h to 15 h.

[0011] Preferably, the molar ratio of the intermediate product to trimethyl orthoformate is 1:1 to 3.

[0012] Preferably, the conditions for the oxidation reaction are: stirring and reacting at 50 °C to 55 °C for 4 h to 5 h.

[0013] Preferably, the mass percentage of the H2O2 solution is 30 wt%, and the volume ratio of the concentration of cisplatin to the H2O2 solution is 0.67 mmol: 12 mL to 14 mL.

[0014] Preferably, the conditions for the esterification reaction are: stirring and reacting at 60 °C to 65 °C for 20 h to 24 h.

[0015] Preferably, the molar ratio of the tetravalent cisplatin product to norcantharidin is 1:2 to 3.

[0016] Preferably, n in the carboxymethyl chitosan solution -NH2 and n in the glutaraldehyde solution -CHO The ratio is 1:0.8 to 2.4. Further, n in the carboxymethyl chitosan solution -NH2 and n in the glutaraldehyde solution -CHO The ratio is 1:1.6.

[0017] Preferably, the volume ratio of the carboxymethyl chitosan solution to the oily orthoester is 10:1 to 5. Further, the volume ratio of the carboxymethyl chitosan solution to the oily orthoester is 10:3.

[0018] Preferably, the molar ratio of the cisplatin prodrug to the photothermal agent indocyanine green is 25 to 29:1. Further, the molar ratio of the cisplatin prodrug to the photothermal agent indocyanine green is 27:1.

[0019] The present invention also protects an injectable organic composite hydrogel for combined photothermal chemotherapy prepared by the above preparation method.

[0020] Preferably, the injectable organic composite hydrogel for combined photothermal chemotherapy is composed of an orthoester complex and an aqueous hydrogel. The orthoester complex is emulsified and uniformly dispersed in the hydrogel, and the hydrogel is cross-linked by carboxymethyl chitosan and glutaraldehyde; wherein, the drugs loaded on the orthoester complex are composed of the chemotherapy drug cisplatin prodrug and the photothermal agent indocyanine green.

[0021] The present invention also protects the application of the injectable organic composite hydrogel for combined photothermal chemotherapy in the preparation of drug carriers, photothermal therapy drugs or anti-cancer drugs.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are: 1. The present invention provides an injectable organic composite hydrogel for combined photothermal chemotherapy, which consists of a hydrogel in the aqueous phase and an oily orthoester dissolved with the chemotherapeutic drug cisplatin prodrug and the photothermal agent indocyanine green. The orthoester complex is emulsified and uniformly dispersed in the hydrogel. The hydrogel is formed by crosslinking the amino group on carboxymethyl chitosan with the aldehyde group on small molecule glutaraldehyde, and dynamic Schiff base imine bonds are formed. The injectable organic composite hydrogel for combined photothermal chemotherapy provided by the present invention can dissolve the two drugs by using the oily orthoester, and then uniformly load them in the hydrogel through emulsification. The dynamic imine bonds of the hydrogel break in response to the tumor microacidic environment, and the orthoester complex successfully escapes. Due to the low specific heat capacity, lipophilicity and pH responsiveness of the oily orthoester, the oily orthoester not only assists in enhancing the photothermal therapy effect of the photothermal agent indocyanine green, but also enhances the penetration of the two drugs in tumor tissues. Along with the degradation of the oily orthoester, the two drugs self-assemble into nanoparticles through hydrogen bonding, enhancing the enrichment and retention ability of the drugs at the tumor site and improving the overall therapeutic effect.

[0023] 2. The injectable organic composite hydrogel for combined photothermal chemotherapy of the present invention has good tissue adhesiveness, injectability, biodegradability, biosafety and photothermal performance, can pass through a syringe smoothly, and is used for the combined treatment of long-term chemotherapy and photothermal therapy of tumors in situ minimally invasively, and significantly improves the therapeutic effect.

[0024] 3. The introduction of the oily orthoester in the present invention not only improves the photothermal conversion efficiency and photothermal stability of the photothermal agent, but also its lipophilicity and acid sensitivity not only enhance the penetration of the two drugs at the tumor site, but also respond to the tumor microacidic environment and degrade, and at the same time assist the two drugs to self-assemble into nanoparticles through hydrogen bonding.

[0025] 4. The present invention uses the combined treatment of photothermal therapy and chemotherapy, which has the effects of synergistic anti-tumor, reducing systemic toxicity and avoiding multi-drug resistance, and has the characteristics of low toxicity, high retention and high efficacy, overcoming the limitations of single treatment. Generally speaking, the injectable organic composite hydrogel for combined photothermal chemotherapy of the present invention solves the problems of difficult drug release, poor enrichment and retention ability of drugs at the tumor site, strong toxic and side effects, and poor therapeutic effect existing in the existing organic composite hydrogels.

[0026] In the present invention, Gel represents an injectable hydrogel, FOE represents an oily orthoester, Pt(IV)-1 represents a cisplatin prodrug, ICG represents indocyanine green, IP represents a mixture of a cisplatin prodrug and indocyanine green, CMCS represents carboxymethyl chitosan, GA represents glutaraldehyde, Gel / FOE represents an injectable organic composite hydrogel, FOE / IP represents an orthoester complex, Gel / FOE / IP represents an injectable organic composite hydrogel for combined photothermal chemotherapy, Gel / IP represents a drug-loaded injectable hydrogel, Gel / FOE / Pt(IV)-1 represents a cisplatin prodrug-loaded injectable organic composite hydrogel, Gel / FOE / ICG represents an indocyanine green injectable organic composite hydrogel, and NIR is near-infrared light at 808 nm. Description of the Drawings

[0027] Figure 1 In the figure, (a) is the SEM image of Gel-1, Gel, and Gel-2, and the inset is the corresponding macroscopic gelation image; (b) is the single-frequency scanning image of Gel-1, Gel, and Gel-2; (c) is the crosslinking degree image of Gel-1, Gel, and Gel-2.

[0028] Figure 2 is the SEM image of Gel / FOE-1, Gel / FOE, and Gel / FOE-2. The upper left inset is the corresponding macroscopic state image, and the lower left inset is the microsection image.

[0029] Figure 3 In the figure, (a) is the Fourier transform infrared spectroscopy image of GA, CMCS, Gel, FOE, and Gel / FOE; (b) is the X-ray diffraction image of GA, CMCS, Gel, Gel / FOE, FOE / IP, and Gel / FOE / IP of Example 1.

[0030] Figure 4 In the figure, (a) is the time scanning image of Gel and Gel / FOE; (b) is the shear rate scanning image of Gel / FOE, and the inset is the needle-passing image of Gel / FOE; (c) is the storage modulus and loss modulus image of Gel / FOE under amplitude scanning; (d) is the storage modulus and loss modulus image of Gel / FOE under high and low strain alternating scanning, and the inset is the self-healing process image.

[0031] Figure 5 In the figure, (a) is H2O / ICG, Gel / ICG, FOE / ICG, Gel / FOE / ICG at a power density of 0.5 W / cm 2Temperature change curve after 5 min of continuous laser irradiation; (b) Infrared imaging diagrams of H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG during the heating process; (c) Temperature change curves of H2O / ICG, Gel / ICG, FOE / ICG, and Gel / FOE / ICG in 4 cycles after 5 min of continuous laser irradiation at a power density of 0.5 W / cm 2 ; (d) Fluorescence intensity change curves of H2O / ICG and FOE / ICG.

[0032] Figure 6 Among them, (a) Diagram of in vitro degradation of Gel / FOE at different pH values; (b) Diagram of in vitro degradation of Gel, Gel / FOE, and Gel / FOE / ICG + NIR in a PBS environment of 0.2 mol / L and pH 6.8.

[0033] Figure 7 Diagram of the escape process of FOE in Gel / FOE at different times.

[0034] Figure 8 Among them, (a) Verification diagram of the degradation of FOE in Gel / FOE after standing for 3 h, 6 h, and 12 h after gelation; (b) Verification diagram of the escape of FOE from Gel in Gel / FOE.

[0035] Figure 9 Among them, (a) Particle size diagram of nanoparticles, where the inset is a TEM diagram; (b) Particle size stability diagram of nanoparticles at different pH values; (c) Potential diagram of nanoparticles at different pH values.

[0036] Figure 10 Drug release diagrams of Pt in Gel / IP and Gel / FOE / IP of Example 1 at different pH values.

[0037] Figure 11 Toxicity diagrams of Gel / IP, Gel / FOE / IP of Example 1, Gel / IP + NIR, and Gel / FOE / IP + NIR containing different concentration gradients of cisplatin co-incubated with HepG2 cells for 48 h.

[0038] Figure 12 Among them, (a) Qualitative uptake diagram of HepG2 cells co-incubated with Gel / IP and Gel / FOE / IP of Example 1 for 4 h, and (b) Quantitative uptake diagram of HepG2 cells co-incubated with Gel / IP and Gel / FOE / IP of Example 1 for 4 h.

[0039] Figure 13Among them, (a) is the tissue adhesion diagram of Gel / FOE to pig skin, pork, and pig liver; (b) is the adhesion diagram of Gel / FOE to glass, plastic, rubber, and metal, and (c) is the adhesion diagram of Gel / FOE / ICG in mice.

[0040] Figure 14 It is the hemolysis schematic diagram of Gel / FOE / Pt(IV)-1.

[0041] Figure 15 Among them, (a) is the tumor volume change diagram of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR of Example 1, and the blank control group of mice; (b) is the tumor weight diagram of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR of Example 1, and the blank control group of mice; (c) is the tumor picture of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR of Example 1, and the blank control group of mice; (d) is the weight change of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR of Example 1, and the blank control group of mice. Detailed implementation manners

[0042] The following combines the drawings to describe the detailed implementation manners of the present invention in detail, but it should be understood that the protection scope of the present invention is not limited by the detailed implementation manners. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0043] Mix the CMCS solution with a mass fraction of 2% and the GA solution with a mass fraction of 5%. The ratio of n -NH2 in CMCS to n -CHO in GA is 1:0.8 to obtain an injectable hydrogel, denoted as Gel-1.

[0044] Mix the CMCS solution with a mass fraction of 2% and the GA solution with a mass fraction of 5%. The ratio of n -NH2 in CMCS to n -CHO in GA is 1:1.6 to obtain an injectable hydrogel, denoted as Gel.

[0045] Mix the CMCS solution with a mass fraction of 2% and the GA solution with a mass fraction of 5%. The ratio of n -NH2 in CMCS to n-CHO The ratio is 1:2.4 to obtain an injectable hydrogel, denoted as Gel-2.

[0046] In the present invention, Gel-1, Gel and Gel-2 were first used for screening injectable hydrogels. The results showed that Gel had the best performance. Then, Gel / FOE-1, Gel / FOE and Gel / FOE-2 were respectively prepared using Gel to screen injectable organic composite hydrogels. Gel / FOE-1, Gel / FOE and Gel / FOE-2 were prepared according to the following steps: Add a 2% CMCS solution to FOE, vortex to make FOE emulsify evenly, and add a 5% GA solution while vortexing. The n in CMCS -NH2 and the n in GA -CHO The ratio is 1:1.6, and V CMCS :V FOE is 10:1 to obtain an injectable organic composite hydrogel, denoted as Gel / FOE-1.

[0047] Add a 2% CMCS solution to FOE, vortex to make FOE emulsify evenly, and add a 5% GA solution while vortexing. The n in CMCS -NH2 and the n in GA -CHO The ratio is 1:1.6, and V CMCS :V FOE is 10:3 to obtain an injectable organic composite hydrogel, denoted as Gel / FOE.

[0048] Add a 2% CMCS solution to FOE, vortex to make FOE emulsify evenly, and add a 5% GA solution while vortexing. The n in CMCS -NH2 and the n in GA -CHO The ratio is 1:1.6, and V CMCS :V FOE is 2:1 to obtain an injectable organic composite hydrogel, denoted as Gel / FOE-2.

[0049] Next, the technical solution of the present invention was further studied using examples. The specific research methods and results are as follows: Example 1 A preparation method of an injectable organic composite hydrogel for combined photothermal chemotherapy includes the following steps: S1. Under a nitrogen atmosphere, add 3-amino-1,2-propanediol and ethyl trifluoroacetate to a dry 500 mL three-necked round-bottom reaction flask containing a magnetic rotor. The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:2. Then add 150 mL of anhydrous acetonitrile as the reaction solvent, and stir the reaction for 12 h while passing nitrogen. After the reaction is completed, rotary evaporate to remove the solvent anhydrous acetonitrile, and dissolve it with 150 mL of ethyl acetate solution containing 100 μL of triethylamine. Then extract it once alternately with 0.5 mol / L potassium bisulfate solution and saturated sodium chloride solution, and dry it with anhydrous magnesium sulfate for 6 h. Rotary evaporate to remove ethyl acetate, and collect the intermediate product.

[0050] Place the intermediate product and trimethyl orthoformate together in a 500 mL dry reaction flask. The molar ratio of the intermediate product to trimethyl orthoformate is 1:2. Add 150 mL of anhydrous acetonitrile as the reaction solvent, add the catalyst p-toluenesulfonic acid monohydrate, and stir the reaction for 12 h while passing nitrogen. Then add triethylamine to terminate the reaction. Rotary evaporate to remove anhydrous acetonitrile, dissolve it with ethyl acetate, and extract it once with saturated sodium bicarbonate and saturated sodium chloride respectively. Collect the organic phase, and rotary evaporate to remove the organic solvent ethyl acetate to obtain FOE.

[0051] S2. Add 200 mg (0.67 mmol) of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve cisplatin. Dropwise add 13 mL of 30 wt% H2O2 solution through a separatory funnel, and stir the reaction for 5 h in the dark at 55 °C. After the reaction is completed, rotary evaporate to remove most of the water and H2O2 solution, and then freeze-dry to obtain a yellow tetravalent cisplatin product.

[0052] Mix 100 mg (0.3 mmol) of the tetravalent cisplatin product and 151 mg (0.9 mmol) of norcantharidin, then dissolve them with 5 mL of anhydrous N,N-dimethylformamide, add them to a round-bottom reaction flask, and then add 121.16 mg (1.2 mmol) of the catalyst triethylamine. Under nitrogen protection, react in the dark at 65 °C for 24 h. After the reaction is completed, distill off N,N-dimethylformamide under reduced pressure, precipitate with anhydrous ether under ice bath, wash the precipitate with acetone, and finally dry it to obtain a pale yellow Pt(IV)-1 powder.

[0053] S3. Place 7.3 mg of Pt(IV)-1 and 0.33 mg of ICG together in 300 μL of FOE, and ultrasonicate for 30 min to completely dissolve the solid to obtain a proester composite, denoted as FOE / IP. Add a 2% CMCS solution to FOE / IP, vortex to emulsify the proester composite evenly, and add a 5% GA solution while vortexing. The ratio of n in CMCS -NH2 to n in GA -CHO is 1:1.6, and V CMCS:V FOE The ratio is 10:3 to obtain an injectable organic composite hydrogel for combined photothermal chemotherapy, denoted as Gel / FOE / IP.

[0054] Example 2 A preparation method of an injectable organic composite hydrogel for combined photothermal chemotherapy, comprising the following steps: S1. Under a nitrogen atmosphere, add 3-amino-1,2-propanediol and ethyl trifluoroacetate to a dry 500 mL three-necked round-bottom reaction flask containing a magnetic rotor. The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1. Then add 150 mL of anhydrous acetonitrile as the reaction solvent, and stir the reaction for 10 h while passing nitrogen; after the reaction is completed, rotary evaporate to remove the solvent anhydrous acetonitrile, and dissolve it with 150 mL of ethyl acetate solution containing 80 μL of triethylamine. Then extract it once alternately with 0.5 mol / L potassium bisulfate solution and saturated sodium chloride solution, dry it with anhydrous magnesium sulfate for 6 h, rotary evaporate to remove ethyl acetate, and collect the intermediate product.

[0055] Place the intermediate product and trimethyl orthoformate together in a 500 mL dry reaction flask. The molar ratio of the intermediate product to trimethyl orthoformate is 1:3. Add 150 mL of anhydrous acetonitrile as the reaction solvent, add the catalyst p-toluenesulfonic acid monohydrate, stir the reaction for 15 h while passing nitrogen, and then add triethylamine to terminate the reaction; rotary evaporate to remove anhydrous acetonitrile, dissolve it with ethyl acetate, extract it once with saturated sodium bicarbonate and saturated sodium chloride respectively, collect the organic phase, rotary evaporate to remove the organic solvent ethyl acetate, and obtain FOE.

[0056] S2. Add 200 mg (0.67 mmol) of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve cisplatin. Dropwise add 12 mL of 30 wt% H2O2 solution through a separatory funnel, and stir the reaction for 4.5 h in the dark at 50 °C. After the reaction is completed, rotary evaporate to remove most of the water and H2O2 solution, then wash it with deionized water and freeze-dry to obtain a yellow tetravalent cisplatin product.

[0057] Mix 100 mg (0.3 mmol) of the tetravalent cisplatin product and 181.2 mg (0.6 mmol) of norcantharidin, then dissolve it with 5 mL of anhydrous N,N-dimethylformamide, add it to a round-bottom reaction flask, and then add 121.16 mg (1.2 mmol) of the catalyst triethylamine. React in the dark at 60 °C under nitrogen protection for 22 h; after the reaction is completed, distill off N,N-dimethylformamide under reduced pressure, precipitate it with anhydrous ether under ice bath, wash the precipitate with acetone, and finally dry it to obtain a pale yellow Pt(IV)-1 powder.

[0058] S3. Place 6.76 mg of Pt(IV)-1 and 0.33 mg of ICG together in 300 μL of FOE, and ultrasonicate for 30 min to completely dissolve the solid, obtaining a proester composite; add a 2% CMCS solution to the proester composite, vortex to emulsify the proester composite evenly, and while vortexing, add a 5% GA solution. The ratio of n -NH2 in CMCS to n -CHO in GA is 1:0.8, and V CMCS :V FOE is 10:5, obtaining an injectable organic composite hydrogel for combined photothermal chemotherapy.

[0059] Example 3 A preparation method of an injectable organic composite hydrogel for combined photothermal chemotherapy, comprising the following steps: S1. Under a nitrogen atmosphere, add 3-amino-1,2-propanediol and ethyl trifluoroacetate to a dry 500 mL three-necked round-bottom reaction flask containing a magnetic rotor. The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:3, then add 150 mL of anhydrous acetonitrile as the reaction solvent, and stir the reaction under nitrogen for 15 h; after the reaction is completed, rotary evaporate to remove the solvent anhydrous acetonitrile, dissolve it with 150 mL of ethyl acetate solution containing 150 μL of triethylamine, and then extract it once with 0.5 mol / L potassium bisulfate solution and saturated sodium chloride solution alternately, and dry it with anhydrous magnesium sulfate for 6 h, rotary evaporate to remove ethyl acetate, and collect the intermediate product.

[0060] Place the intermediate product and trimethyl orthoformate together in a 500 mL dry reaction flask. The molar ratio of the intermediate product to trimethyl orthoformate is 1:1. Add 150 mL of anhydrous acetonitrile as the reaction solvent, add the catalyst p-toluenesulfonic acid monohydrate, stir the reaction under nitrogen for 10 h, and then add triethylamine to terminate the reaction; rotary evaporate to remove anhydrous acetonitrile, dissolve it with ethyl acetate, and then extract it once with saturated sodium bicarbonate and saturated sodium chloride respectively, collect the organic phase, rotary evaporate to remove the organic solvent ethyl acetate, and obtain FOE.

[0061] S2. Add 200 mg (0.67 mmol) of cisplatin to a 100 mL round-bottom flask, then add 12 mL of deionized water to dissolve cisplatin, and dropwise add 14 mL of 30 wt% H2O2 solution through a separatory funnel, and stir the reaction in the dark at 50 °C for 5 h. After the reaction is completed, rotary evaporate to remove most of the water and H2O2 solution, then wash with deionized water and freeze-dry to obtain a yellow tetravalent cisplatin product.

[0062] 100 mg (0.3 mmol) of the tetravalent cisplatin product and 120.8 mg (0.6 mmol) of norcantharidin were mixed, then dissolved in 5 mL of anhydrous N,N-dimethylformamide, added to a round-bottom reaction flask, and then 121.16 mg (1.2 mmol) of the catalyst triethylamine was added. Under nitrogen protection, the reaction was carried out at 60 °C in the dark for 24 h. After the reaction, N,N-dimethylformamide was removed by distillation under reduced pressure. Precipitation was carried out with anhydrous diethyl ether under an ice bath, and the precipitate was washed with acetone and finally dried to obtain light yellow Pt(IV)-1 powder.

[0063] S3: 7.84 mg of Pt(IV)-1 and 0.33 mg of ICG were placed together in 300 μL of FOE, and ultrasonicated for 30 min to completely dissolve the solid to obtain a proester composite. A 2% CMCS solution was added to the proester composite, and vortexed to emulsify the proester composite evenly. While vortexing, a 5% GA solution was added. The ratio of n in CMCS -NH2 to n in GA -CHO was 1:2.4, and V CMCS :V FOE was 10:1 to obtain an injectable organic composite hydrogel for combined photothermal chemotherapy.

[0064] In Examples 1 to 3 of the present invention, injectable organic composite hydrogels for combined photothermal chemotherapy with excellent performance were all prepared. Below, taking Gel-1, Gel, Gel-2, Gel / FOE-1, Gel / FOE, Gel / FOE-2 and Gel / FOE / IP of Example 1 as examples, the research was carried out. The specific research methods and results are as follows: I. Determination and analysis of injectable hydrogels and injectable organic composite hydrogels: 1. Screening of hydrogels: Macroscopic morphological structure observation: After the injectable hydrogel and injectable organic composite hydrogel are prepared, take pictures with a camera and record the macroscopic state. Microscopic morphological structure observation: After Gel / FOE-1, Gel / FOE and Gel / FOE-2 are prepared, take them out, cut them into thin slices with a blade, place them under a microscope for observation and take pictures for recording. Scanning electron microscope sample preparation: Use liquid nitrogen to quickly freeze the injectable hydrogel and injectable organic composite hydrogel, then perform brittle fracture treatment, freeze-dry, paste the conductive tape on the surface of the silicon wafer, cut a part of the brittle fracture surface of the completely freeze-dried injectable hydrogel and injectable organic composite hydrogel and adhere it to the conductive tape, then place the silicon wafer with the hydrogel sample on the edge of the sample stage, perform gold spraying treatment on the injectable hydrogel and injectable organic composite hydrogel, and observe with SEM. Single frequency scanning: Use a Kinexus Lab+ rotational rheometer to scan Gel-1, Gel and Gel-2 to investigate the change of a single frequency. Crosslinking degree determination: Utilize the reaction between the amino group of CMCS and the aldehyde group of GA to generate an imine bond and a molecule of water at the same time to determine the crosslinking degree of Gel-1, Gel and Gel-2.

[0065] 2. Fourier transform infrared spectroscopy and X-ray diffraction determination of hydrogels: Thoroughly mix the freeze-dried Gel, freeze-dried Gel / FOE, and CMCS powder with potassium bromide powder respectively, grind them into fine powder, and press them into transparent thin slices; for the liquid samples GA and FOE, press potassium bromide powder into a thin slice, then evenly smear them on its surface respectively, and then dry them with a mercury lamp. Scan in the wavenumber range of 500 cm -1 ~4000 cm -1 with a Fourier transform infrared spectrometer. Load the freeze-dried Gel, freeze-dried Gel / FOE, freeze-dried Gel / FOE / IP, and CMCS powder into the grooves of the glass slides respectively, and scrape the surface flat; directly load the liquid samples GA and FOE / IP into the grooves of the glass slides and put them into an X-ray diffractometer for structural characterization.

[0066] 3. Rheological property determination of hydrogels: Use a Kinexus Lab+ rheometer to evaluate the rheological properties of Gel and Gel / FOE. First, under the condition of constant stress, perform frequency scanning on Gel and Gel / FOE, and the scanning range is 0.1 Hz to 10 Hz to monitor the changes of the storage modulus G' and the loss modulus G”. Subsequently, perform amplitude scanning on Gel / FOE, and the scanning range is 0.1% to 1000% to determine the linear viscoelastic region at a fixed frequency of 1 Hz. Next, quantitatively investigate the self-healing ability of Gel / FOE through dynamic oscillatory rheology tests at a constant frequency of 1 Hz. Finally, analyze the change of viscosity to evaluate the shear thinning behavior of Gel / FOE at different shear rates, and the shear rate range is 0.1 s -1~100 s -1 。

[0067] 4. Photothermal heating, photothermal stability, and fluorescence intensity measurement: Different groups containing 250 μg / mL of ICG were prepared, specifically H2O / ICG, FOE / ICG, Gel / ICG, Gel / FOE / ICG. In Gel / FOE / ICG, the volume of Gel was 1052 μL and the volume of FOE was 300 μL. They were placed in centrifuge tubes respectively and irradiated with an 808 laser at 0.5 W / cm 2 for 5 min. During the irradiation process, thermosensitive photos were taken with an infrared thermal imager and the temperature was recorded. The solutions of the above different groups were freshly prepared and irradiated with an 808 nm laser at 0.5 W / cm 2 for 5 min. After the temperature of each group dropped to room temperature, the fluorescence intensities of the H2O / ICG and FOE / ICG groups were measured using a microplate reader. The above operations were repeated four times and the data were recorded to observe the photothermal stability of each group and the effect of FOE on the fluorescence intensity of ICG.

[0068] 5. Determination of in vitro degradation: After the Gel / FOE was prepared, it was weighed and denoted as M0. It was placed in PBS solutions with different pH values, specifically PBS solutions with pH 7.4, pH 6.8, pH 5.0, and pH 5.0 / GSH. pH 5.0 / GSH means an environment with glutathione present under the condition of pH 5.0, and it was oscillated at 37 °C and 100 rpm. Gel, Gel / FOE, and Gel / FOE / ICG+NIR groups were respectively placed in a 0.2 mol / L, pH 6.8 PBS solution. The operation of the Gel / FOE / ICG+NIR group was as follows: Gel / FOE / ICG was irradiated with light once a day. Three parallels were set for each group. At the preset time points, the excess PBS solution was removed, and Gel, Gel / FOE, and Gel / FOE / ICG+NIR were weighed and denoted as Mr. The remaining mass ratio was calculated through the formula

[0069] 6. Verification of the escape of FOE in Gel / FOE: Six prepared Gel / FOE and Gel were each taken and immersed in 5 mL of a 0.2 mol / L, pH 6.8 PBS solution, and placed on a 37 °C constant temperature shaker. At the preset time points of 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, Gel / FOE and Gel were taken, cut into thin slices at the edge and in the center, and observed and photographed under a microscope.

[0070] ​7. Experimental verification of whether FOE degrades in Gel and can escape from Gel: Place Gel / FOE in a 4°C refrigerator. At preset time points of 3 h, 6 h, and 12 h, extract FOE using dichloromethane, remove dichloromethane by rotary evaporation, and detect by 1 1H NMR. Immerse Gel / FOE in a 0.2 mol / L PBS solution with a pH of 6.8. At 1 h, freeze-dry a portion of the buffer of Gel / FOE and detect by 1 1H NMR.

[0071] 8. Particle size, particle size stability, and potential changes of nanoparticles: Place the Gel / FOE / IP of Example 1 in a 0.2 mol / L PBS solution with a pH of 6.8, and place it in a constant temperature shaker at 37°C. After 12 h, take the supernatant, centrifuge it in a centrifuge at 8000 rpm for 5 min to remove macromolecular substances in Gel / FOE / IP to obtain nanoparticles. At room temperature, use DLS to measure the particle size of the nanoparticles, the potential changes in different environments with pH values of 7.4, 6.8, and 5.0 respectively, and the particle size stability in different environments with pH values of 7.4, 6.8, 5.0, and 5.0 / GSH respectively.

[0072] 9. Drug release of cisplatin prodrug: Seal Gel / IP and Gel / FOE / IP in dialysis bags with a molecular weight cut-off of 14,000 Da, and then immerse them in 5 mL of PBS solutions with different ratios of 0.2 mol / L. The pH values of the PBS solutions are 7.4, 6.8, 5.0, and 5.0 / GSH respectively. Set three parallels for each group. At preset time points of 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h, take out the PBS solution and add 5 mL of new PBS solution. The obtained samples are measured by high performance liquid chromatography. The preparation method of Gel / IP is as follows: Mix 7.3 mg of Pt(IV)-1 and 0.33 mg of ICG in a 2% CMCS solution by mass fraction, vortex, and add a 5% GA solution while vortexing. The n -NH2 in CMCS and the n -CHO in GA have a ratio of 1:1.6.

[0073] Result analysis: 1. Screening of injectable hydrogels: As shown in Figure (b) of Figure 1 , at the same frequency, G' is always greater than G" for the three groups of injectable hydrogels, indicating that the three groups of injectable hydrogels have reached the gel state. As shown in Figure (c) of Figure 1 , the crosslinking degrees of Gel-1, Gel, and Gel-2 are 56.5%, 77.1%, and 61.4% respectively. Compared with the Gel-1 and Gel-2 groups, the Gel group has higher mechanical strength. ThroughFigure 1 As can be seen from Figure (a) in , the morphology of Gel-1 is rough and the pores are disorderly. The pore structure of the Gel group is uniform, presenting a regular and dense porous network structure. The Gel-2 group shows a flaky stacking as a whole, and the pores are covered. In order to avoid the influence of the addition of FOE on the structure of the injectable hydrogel, a porous network structure with high mechanical strength and regular morphology was selected for subsequent experiments. Therefore, the Gel group was selected for subsequent experiments.

[0074] 2. Screening of the amount of FOE in Gel / FOE: Figure 2 In the Gel / FOE-1 group in , although the SEM image is still a porous structure, through the microscopic observation of the Gel / FOE-1 section, it can be seen that too little FOE is added, which is not conducive to the subsequent drug loading. The Gel / FOE group has a smooth morphology, still maintaining a regular porous network structure, and it can be seen from the microscopic image that FOE is evenly dispersed in Gel, which is conducive to the uniform release of the drug after subsequent drug loading. The Gel / FOE-2 group contains a large amount of FOE, and the SEM image shows that some FOE precipitates on its surface, and the original reticular pore structure is completely destroyed. Considering comprehensively, we selected the Gel / FOE group for subsequent experiments.

[0075] 3. Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) analysis: Figure 3 Figure (a) in shows the FT-IR spectra of GA, CMCS, Gel, FOE, and Gel / FOE in the range of 500 cm -1 ~4000 cm -1 . The stretching vibration absorption peak V -1 at 1722 cm C=O of GA and the stretching vibration absorption peak V -1 at 3442 cm -NH2 of CMCS disappear in Gel, and a V -1 at 1624 cm C=N appears in Gel, indicating that after the formation of the Gel three-dimensional network structure, the aldehyde group and amino group are consumed, forming a Schiff base dynamic cross-linking structure. In Gel / FOE, the characteristic peak V -1 of FOE appears at 1070 cm C-F , and the stretching vibration absorption peak V -1 at 1624 cm C=N still appears, indicating that FOE is successfully added and does not destroy the cross-linking structure of Gel.

[0076] XRD analyzes the sample structure through the X-ray crystal diffraction effect. As shown in Figure 3As shown in Figure (b), CMCS shows a broad peak at 20.8°, GA shows a broad diffraction peak at 23.71°, and after the formation of Gel, a broad peak appears at 22.3°. This indicates that CMCS and GA have crosslinked, changing the crystal structures of CMCS and GA, proving that Gel has been successfully prepared. At the same time, the broad peak positions of Gel / FOE / IP are the same as those of Gel / FOE and FOE / IP, proving the correct structure of Gel / FOE / IP.

[0077] 4. Rheological property analysis of Gel / FOE: As Figure 4 shown, Figure 4 Figure (a) in it shows that the storage modulus G’ of Gel and Gel / FOE is greater than the loss modulus G”, indicating that Gel and Gel / FOE always maintain a crosslinked structure and have good stability. However, the G’ and G” of Gel / FOE are lower than those of Gel, indicating that the addition of FOE reduces the mechanical strength of Gel to a certain extent. Figure 4 Figure (b) in it shows that the Schiff base crosslinked structure of Gel / FOE is damaged under shear action. As the shear rate continuously increases, the viscosity of Gel / FOE rapidly decreases and finally remains constant, proving that Gel / FOE has good injectability. The inset in Figure (b) also macroscopically proves the injectability of Gel / FOE. Figure 4 Figure (c) in it determines the critical strain values of G’ and G” of Gel / FOE, and based on this value, high-strain and low-strain scans in Figure (d) are carried out. Figure 4 Figure (d) in it shows that under alternating high and low strain scans, Gel / FOE realizes the transformation between the gel state and the sol state. At high strain, G’ of Gel / FOE is less than G”, and at this time Gel / FOE is in the sol state; while at low strain, G’ is greater than G”, and Gel / FOE returns to the gel state. This sol-gel transformation process can be repeated, thus proving that Gel / FOE has self-healing properties. This self-healing property is endowed by the dynamic imine bonds formed between amino groups and aldehyde groups. The inset in Figure (d) macroscopically proves the good self-healing properties of Gel / FOE.

[0078] 5. Study on photothermal conversion performance and stability: As Figure 5 shown in Figure (a) and Figure (b), after FOE / ICG is irradiated with a 0.5 W / cm 2 laser for 5 minutes, the temperature can reach 74.3 °C, while the temperature of H2O / ICG is only 47.2 °C after 5 minutes of irradiation, indicating that FOE with a low specific heat capacity is beneficial to improving the photothermal efficiency of ICG. The Gel / ICG and Gel / FOE / ICG groups finally reach 48.7 °C and 54 °C respectively, indicating that loading FOE in Gel is beneficial to improving the overall photothermal temperature rise. AsFigure 5 As shown in Figures (c) and (d) therein, the FOE / ICG group still maintained a high photothermal effect after multiple cycles of heating, while the photothermal ability of H2O / ICG decreased significantly under the same conditions. Compared with the Gel / ICG group, the Gel / FOE / ICG group showed better photothermal stability. At the same time, after multiple cycles of heating, the FOE / ICG group could still maintain a high fluorescence intensity, indicating that FOE improved the structural stability of ICG molecules, which would be more conducive to the role of ICG in subsequent cancer treatment.

[0079] 6. Study on in vitro degradation: The degradability of Gel / FOE plays an important role in stable drug release. As Figure 6 shown, the degradation rate of Gel / FOE was relatively fast within 48 h. At 48 h, the remaining mass ratios in the pH 7.4, pH 6.8, pH 5.0, and pH 5.0 / GSH environments were 39.3%, 35.0%, 15.7%, and 14.7%, respectively. The results showed that a decrease in pH would accelerate the cleavage of the dynamic imine bonds in Gel / FOE to accelerate the disintegration of Gel / FOE. In the pH 6.8 environment, the degradation rate of Gel / FOE was faster than that of the Gel group because the dynamic imine bond was acid-sensitive, and the cleavage of the imine bond in the slightly acidic environment and the escape of FOE accelerated the overall degradation rate. The Gel / FOE / ICG + NIR group had the fastest degradation rate, indicating that photothermal treatment was beneficial to the disintegration of Gel / FOE.

[0080] 7. Verification of the escape process of FOE from Gel / FOE: As Figure 7 shown, at 0 h, the FOE in the edge and central parts of Gel / FOE was uniformly dispersed in the form of droplets. As time passed, compared with Gel, the number of FOE in the edge Gel / FOE gradually decreased, and the droplets gradually became smaller, and almost completely escaped at 24 h. The size and number of the central FOE droplets hardly changed within 24 h. The results showed that the escape process of FOE was from the edge to the center of Gel / FOE, and the droplets themselves escaped from the outside to the inside.

[0081] 8. Experimental verification of whether FOE degrades in Gel and can escape from Gel: Whether FOE degrades in Gel and whether FOE can escape from Gel are the keys to tumor treatment. As Figure 8 seen, within 12 h, the characteristic peak and degradation peak of FOE in Gel did not change significantly, proving that FOE did not degrade in Gel within 12 h. Partially degraded FOE was detected in the PBS solution, proving that FOE could successfully escape from Gel and could degrade in a slightly acidic environment.

[0082] 9. Particle size, particle size stability, and potential changes of nanoparticles: AsFigure 9 As shown in Figure (a) in Figure 9 As shown in Figure (b) in Figure 9 As shown in Figure (c) in

[0083] 10. Drug release of cisplatin: As Figure 10 shown, under the conditions of pH 7.4, pH 6.8, and pH 5.0, the cisplatin drug release amounts in the Gel and Gel / FOE groups were less than 20% of the total drug amount. However, under the condition of pH 5.0 / GSH, the cisplatin drug release amounts reached 58.8% and 70.3% of the total drug amount respectively. This is because under the stimulation of pH 5.0 / GSH, tetravalent cisplatin was reduced to divalent cisplatin, the nanostructure was destroyed, and cisplatin was released. The presence of FOE accelerated the effective escape of the drug from Gel.

[0084] II. In vitro application experiments: 1. Cytotoxicity evaluation: Toxicity experiment on HepG2 cells: After discarding the upper layer of the culture medium using the cultured cells, PBS buffer solution was added to rinse the adherent cells, trypsin digestion solution was added for digestion, centrifuged, and after resuspending the cells, a cell suspension was obtained. The 96-well plates were sterilized before use. 180 μL of the cell suspension was aspirated, with a density reaching 4000 cells / well. After overnight culture to make them adhere, subsequent operations could be carried out. In this experimental group, there were Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR, negative control group, and positive control group. Each group had 6 parallel sets. The negative control group was the culture medium, and the positive control group was the culture medium + cells; among them, in Gel / IP, Gel / IP+NIR, Gel / FOE / IP, and Gel / FOE / IP+NIR, the concentration gradients of cisplatin were all 4.1 μmol / L, 8.2 μmol / L, 16.4 μmol / L, 32.8 μmol / L, and 65.6 μmol / L. According to the group requirements, the plates were paved. After the cells adhered, the drugs of each concentration gradient experimental group were added. The non-irradiated group was incubated for 48 h. The 96-well plates of the irradiated group were taken out at 24 h, and an 808 nm laser was used at 0.5 W / cm 2Irradiate with light from below for 5 min. After the irradiation ends, continue the incubation for 24 h. After the incubation ends, discard the culture medium and the remaining hydrogel. Add 180 μL of fresh culture medium and 20 μL of thiazolyl blue solution to each well, and incubate together for 4 h. After the incubation ends, discard the upper-layer culture medium. Add 150 μL of dimethyl sulfoxide to each well, place it in a constant-temperature shaker at 37 °C for reaction for 20 min. After the reaction ends, use an enzyme-linked immunosorbent assay (ELISA) reader to detect and record the absorbance value of each well at a UV wavelength of 570 nm.

[0085] 2. Qualitative and quantitative uptake of cells: Qualitative uptake: Take the cultured adherent cells, digest them, and prepare a cell suspension for standby. Place a sterile coverslip in a six-well plate, add 1.8 mL of the cell suspension, and the cell density is about 1×10 5 cells / well. Incubate overnight to allow the cells to adhere to the wall. Add drugs Gel / IP and Gel / FOE / IP to it, and the cisplatin concentration of both is 65.6 μmol / L. Co-culture for 4 h. After the culture ends, remove the remaining hydrogel and culture medium, wash the coverslip thoroughly with PBS buffer solution, fix the cells with 4 wt% paraformaldehyde solution for 10 min, then use PBS buffer solution to remove the residual fixing solution, discard the excess PBS buffer solution, add DAPI staining agent to stain the cell nuclei for 10 min, use PBS buffer solution to wash away the excess staining agent, and then observe and photograph under a laser confocal microscope.

[0086] Quantitative uptake: After the cells are digested, plated, cultured, and adhered, add drugs Gel / IP and Gel / FOE / IP to it, and the cisplatin concentration of both is 65.6 μmol / L. There are three parallel controls in each group. After co-culture for 4 h, remove the remaining hydrogel and culture medium, wash the cells three times with PBS buffer solution, discard the excess PBS buffer solution, add 200 μL of trichloroacetic acid lysate to each well, shake well, place it in a refrigerator at 4 °C for 30 min, collect the cells in the wells, centrifuge at 1.2×10 4 rpm for 10 min, aspirate the supernatant and add concentrated nitric acid for digestion, and finally use a spectrometer to detect the drug content to calculate the drug uptake.

[0087] 3. Adhesion detection of Gel / FOE: Place different Gel / FOE on pigskin, pork, and pig liver respectively, and perform stretching, twisting, and squeezing respectively, and observe and photograph. Place different Gel / FOE on glass, plastic, rubber, and metal respectively, and observe and photograph the adhesion of Gel / FOE to glass, plastic, rubber, and metal. Subcutaneously inject Gel / FOE into mice, and dissect and observe and photograph after 10 min.

[0088] 4. Hemolysis experiment: Draw 500 μL of blood from the hearts of mice into a centrifuge tube containing sodium heparin, mix quickly, then add 5 mL of PBS buffer solution, centrifuge at 2500 rpm for 5 min, discard the supernatant, repeat the washing step until the supernatant is clear, centrifuge to collect red blood cells, add 10 mL of normal saline and gently mix with the red blood cells to prepare a red blood cell suspension for subsequent experiments. In this experiment, a positive control group, a negative control group, and experimental groups with different concentration gradients were set up, and three parallel controls were set in each group. The positive control group was a mixture of 500 μL of cell suspension and 500 μL of 0.1% Triton X-100, and the negative control group was a mixture of 500 μL of cell suspension and 500 μL of normal saline. After mixing 500 μL of cell suspension with the materials in each group, incubate them together in a constant temperature incubator at 37 °C for 1 h. After the incubation, centrifuge the samples in each group at 10000 rpm for 5 min, take pictures and record, aspirate the supernatant into a 96-well plate, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance values of each group at a UV wavelength of 570 nm and record the data.

[0089] 。

[0090] Result analysis: 1. Cytotoxicity evaluation: As Figure 11 seen, with the increase of the drug administration concentration in each group, the cytotoxicity to HepG2 cells increased with a certain dose-dependence. Compared with the non-irradiated group, the irradiated group showed higher cytotoxicity, indicating that photothermal treatment had a certain killing effect on tumor cells. The Gel / IP group had certain cytotoxicity because with the degradation of Gel, GA fell off, and the released drug produced toxicity to cells. Gel / FOE / IP had higher cytotoxicity than Gel / IP. On the one hand, due to the oil-water incompatibility, more drugs were released from Gel / FOE. On the other hand, after the degradation of FOE, the two drugs self-assembled into nanoparticles, which were more easily taken up by cells, thus producing stronger cytotoxicity. The results of Gel / FOE / IP + NIR showed that the presence of FOE was beneficial to further improve the combined effect of photothermal therapy and chemotherapy.

[0091] 2. Qualitative and quantitative cell uptake: The uptake of drugs was observed by a laser confocal microscope. As Figure 12 shown in Figure (a) of Figure 12 , obvious red fluorescence appeared in the tumor cells, indicating that ICG was internalized by the cells. At the same time, the fluorescence intensity of Gel / IP was significantly weaker than that of Gel / FOE / IP. To further verify the uptake ability of tumor cells to various drug preparations, we also lysed the cells to detect the platinum content in the cell content extract. As 6, the drug uptake in Gel / FOE / IP is 10.6 μg / 1×10 6 . The qualitative and quantitative uptake results of cells showed that FOE in Gel / FOE encapsulated two drugs. Utilizing the property of immiscibility between oil and water, with the escape of FOE, the escape of the two drugs from Gel / FOE was accelerated. At the same time, after FOE degradation, the two drugs self-assembled to form nanoparticles, which were more easily taken up by cells.

[0092] 3. Adhesion detection of Gel / FOE: From Figure 13 In the tissue adhesion experiment of Gel / FOE, it could firmly adhere to the surfaces of animal epidermis, liver, and muscle tissues. And after operations such as spreading, squeezing, twisting, and stretching on the above tissues, Gel / FOE still firmly adhered to the surface of biological tissues. The adhesion of Gel / FOE was mainly formed through the interaction between covalent bonds and non-covalent bonds. CMCS is a natural polysaccharide derivative with many active groups, which can interact with amino, carboxyl, and hydroxyl groups on the protein surface in biological tissues through hydrogen bonds and imine bonds, thus having good bioadhesion. To verify the injectability and in vivo adhesiveness of Gel / FOE, we injected Gel / FOE subcutaneously into mice. The results showed that Gel / FOE could be injected into the mice and fixed under the skin of the mice, and it was elastic and regular in shape. Therefore, Gel / FOE has good biological tissue adhesion performance and in vivo injectability.

[0093] 4. Hemolysis experiment: Through Figure 14 It was seen that in the positive control group treated with TritonX-100, due to the rupture of the red blood cell membrane, hemoglobin was released, and the supernatant in the centrifuge tube showed red, and there was no red blood cell precipitation at the bottom of the centrifuge tube, and the hemolysis rate was 100%. While there was no obvious difference between different concentrations of Gel / FOE / Pt(IV)-1 and the negative control group. The supernatant was clear, and there was red blood cell precipitation at the bottom of the centrifuge tube. The hemolysis rate of all experimental groups was lower than 5%, proving that there was no obvious hemolysis phenomenon, having high blood compatibility, so the biological safety was good, providing a feasibility guarantee for the following animal experiments. Gel / FOE / Pt(IV)-1 was prepared according to the following steps: Disperse 7.3 mg of Pt(IV)-1 in 300 μL of FOE, then add a 2% CMCS solution, vortex, and while vortexing, add a 5% GA solution. The ratio of n -NH2 in CMCS to n -CHO in GA was 1:1.6, and V CMCS :V FOE was 10:3 to obtain Gel / FOE / Pt(IV)-1.

[0094] III. In vivo application experiment: When the tumor volume reached 200 mm 3 , 42 tumor-bearing mice were randomly divided into 7 groups, namely FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, Gel / FOE / IP+NIR, and the blank control group (the blank control group was the normal saline group). On the 1st, 4th, and 7th days, drugs were injected in situ into the tumors. In the light-irradiated groups, an 808 laser was used to irradiate for 5 minutes at 0.5 W / cm 2 every day for the first seven days. Except for the blank control group, the dosage of cisplatin in each group was 6 mg / kg. The mice were observed for 14 days, and the body weight, tumor volume, and survival status of the mice were recorded every day. On the 14th day, the mice were sacrificed, and the main visceral tissues, including the heart, liver, spleen, lungs, kidneys, and tumors, were dissected and removed. The tumors obtained from the dissection were washed with normal saline and wiped clean, and then weighed and photographed.

[0095] Result analysis: On the 14th day, the tumor volume and tumor mass of the blank control group reached 3069.94 mm 3 and 4.2 g respectively. However, compared with the control group, the tumor growth of FOE / IP, FOE / IP+NIR, Gel / IP, Gel / IP+NIR, Gel / FOE / IP, and Gel / FOE / IP+NIR was relatively slow, and the tumor volume and tumor mass slowly increased to 1000.71 mm 3 and 1.53 g, 114.33 mm 3 and 0.2 g, 1846.21 mm 3 and 3 g, 798.33 mm 3 and 1.1 g, 1046.54 mm 3 and 2.45 g, 123.58 mm 3With 0.43 g, the growth of tumors in mice was inhibited to varying degrees. Compared with the non-irradiated group, the irradiated group had smaller tumor volume and mass, showing a better tumor inhibitory effect. The mortality rates of FOE / IP and FOE / IP+NIR on the 14th day were 33.3% and 66.7% respectively, which was due to the acute toxicity caused by the diffusion of the orthoester complex. The tumor cure rates of Gel / IP+NIR and Gel / FOE / IP+NIR on the 14th day were 16.7% and 50% respectively, and the survival rate of mice was 100%, showing good biosafety and therapeutic ability. The therapeutic effect of the Gel / FOE / IP+NIR group was the most prominent. The reason was that Gel / FOE achieved the enrichment of drugs at the tumor site. During the degradation of Gel / FOE, the escape of FOE accelerated the escape of drugs, improved the photothermal performance, and promoted the diffusion and penetration of drugs at the tumor site. After responding to the microenvironment degradation, it assisted the self-assembly of the two drugs to form nanoparticles, greatly improving the enrichment and retention ability of drugs at the tumor site, reducing the systemic toxic side effects, and improving the therapeutic efficacy.

[0096] Obviously, those skilled in the art can make various changes and deformations to the invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and deformations.

Claims

1. A method for preparing an injectable organic composite hydrogel combined with photothermal chemotherapy, characterized in that: The steps include: Under an inert atmosphere, 3-amino-1,2-propanediol is mixed with ethyl trifluoroacetate, and amidation reaction is carried out in a liquid phase, and the solvent is separated to obtain an intermediate product; the intermediate product is mixed with trimethyl orthoformate, and acetalization reaction is carried out in a liquid phase under catalysis, and the solvent is separated to obtain an oily orthoester; The cisplatin prodrug and the photothermal agent indocyanine green are co-dispersed in an oily orthoester to obtain an orthoester complex, and then a carboxymethyl chitosan solution is added and vortexed to emulsify the orthoester complex evenly. Glutaraldehyde solution is added while vortexing to obtain an injectable organic composite hydrogel combined with photothermal chemotherapy.

2. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The conditions for the amidation reaction are: stirring at room temperature for 10 h to 15 h.

3. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of 3-amino-1,2-propanediol to ethyl trifluoroacetate is 1:1-3.

4. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The conditions for the acetalization reaction are: stirring at room temperature for 10 h to 15 h.

5. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of the intermediate product to trimethyl orthoformate is 1:1-3.

6. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The mass fraction of carboxymethyl chitosan solution is 2%, the mass fraction of glutaraldehyde solution is 5%, and the n -NH2 With glutaraldehyde solution -CHO The ratio is 1:0.8~2.

4.

7. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 6, characterized in that: The volume ratio of carboxymethyl chitosan solution to oily orthoester is 10:1~5.

8. The method for preparing the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 1, characterized in that: The molar ratio of cisplatin prodrug to photothermal agent indocyanine green is 25~29:

1.

9. An injectable organic composite hydrogel combined with photothermal chemotherapy prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the injectable organic composite hydrogel combined with photothermal chemotherapy according to claim 9 in the preparation of drug carriers, photothermal therapeutic drugs or anticancer drugs.

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